: MS is an autoimmune neurodegenerative disease of the central nervous system (CNS) in which the etiology is not well understood. Although, auto-aggressive CD4+ T cells play a central role, the breakdown of immune tolerance mechanisms that permits activation of naive myelin-specific T cells is considered an initial step in the pathogenesis of MS. A number of pivotal studies in rodent models have substantiated that Ag- specific Tregs have a significant role in modulating autoimmune CNS disease and can be highly effective at treating MS. Consequentially, there has been a major focus in developing protocols that stimulate Treg numbers and their function. Unfortunately, successful therapeutic use of Tregs has been limited by the lack of safe and effective Ag-specific protocols for isolation and expansion that are suitable for translation. The AAV gene transfer platform has clearly demonstrated that hepatocyte-restricted transgene expression from an optimized AAV vector can reliably induce immune tolerance to various therapeutic proteins. Importantly, tolerance is dependent on achieving and maintaining adequate hepatocyte-restricted transgene expression that induces Ag-specific CD4+CD25+FoxP3+ Tregs. Recently, we have successfully developed a clinically relevant Adeno-associated Virus (AAV) immunotherapy that is not only capable of preventing the development of EAE, but can also reverse the neurological symptoms of preexisting disease. This vector-based immunotherapy uses the full-length protein coding sequence of a myelin-derived protein, which abrogates the need to identify HLA/MHC specific epitopes, making this unique approach universally applicable. Mechanistically, this process is based on the induction of immunological tolerance mediated by antigen specific Tregs. We hypothesize that a persistent immunological tolerance, independent of MHC restrictions can be established against three immunogenic myelin proteins, simultaneously. That such tolerance is mediated by the induction and expansion of Ag-specific Tregs that can prevent development of and reverse existing disease in the EAE model of MS. Aim 1: Develop, optimize and compare in vivo performance of AAV-PLP and -MBP vectors, and assess functional suppression of Ag-specific Tregs Aim 2: Demonstrate that AAV immunotherapy vectors can prevent or ameliorate disease in genetically diverse strains of mice. Aim3: Determine the minimum effective vector (MEV) dose for abrogation of disease without an adverse immune response or hepatotoxicity. Aim 4: Establish a multi-vector / multi-gene immunotherapy platform.